1. Field of the Invention
This invention relates to the discovery of new bacterial aminopeptidases. More particularly, the invention is directed to an important enzyme activity the deletion or overexpression of which from bacteria improves the respective recovery of uncleaved or cleaved polypeptides produced in the bacteria such as recombinant polypeptides.
2. Description of Related Art
Some proteins have their N-terminal amino acid residue clipped off when they are made in gram-negative bacteria and archaebacteria such as E coli due to the presence of aminopeptidases in the cells. As a result, an impurity closely related to the wild-type polypeptide is introduced into the cell culture either simultaneously or upon subsequent cell lysis as part of the product purification process. This impurity must be removed from the wild-type polypeptide if therapeutically useful proteins are to be prepared. An example is human growth hormone (hGH), which has its N-terminal phenylalanine residue cleaved when made in E. coli. This variant form of hGH (des-phe hGH), produced upon cell lysis to form a mixture with the unclipped hGH (native hGH), is difficult to remove from the mixture. Such removal requires subjection of the mixture to hydrophobic interaction chromatography. It would be desirable to avoid this extra purification step.
Additionally, it is desired in some instances to obtain polypeptides with the N-terminal amino acid residue cleaved and to amplify the quantities of such polypeptides relative to the native-sequence counterpart to obtain purer cleaved material.
Several of the known E. coli aminopeptidases have broad specificity and can cleave a variety of residues at the N-terminus, e.g., pepA, pepB, and pepN (Escherichia coli and Salmonella, Frederick C. Neidhardt (Ed), ASM Press. Chapter 62 by Charles Miller-Protein Degradation and Proteolytic Modification, pp 938-954 (1996); Gonzales and Robert-Baudouy, FEMS Microbiology Reviews. 18 (4):319-44 (1996). The gene yfcK encoding b2324 found in the K12 strain of E. coli was listed as a “putative peptidase” by the E. coli genome sequencing project (Blattner et al., Science, 277: 1453-62 (1997)) in the GenBank database (accession number AE000321), but no further information on its enzyme activity is provided. The homolog in E. coli strain O157:H7 is identical to the yfcK gene in the K12 strain. There is a need in the art to identify bacterial aminopeptidases that can be manipulated to obtain purer uncleaved or cleaved polypeptides.